The rat cultured pineal gland was used to study tetrahydrobiopterin (BH4) regulation. Addition of the BH4 precursor, guanosine to the culture medium had no effect on levels of pineal [BH4 + quinonoid-dihydrobiopterin (qBH2)], [BH2 + biopterin (B)] or medium B measured 24 or 48 hours later. Addition of the putative precursor, sepiapterin increased pineal [BH2 + B] levels and decreased pineal [BH4 + qBH2] levels up to 2-fold and also markedly increased medium B (up to 7-fold) after 48 hours in a concentration-dependent manner. Medium B reflected either BH2 and/or B released from pineals, but did not reflect release of BH4, which was rapidly degraded in medium to pterin and non-fluorescent compounds. Addition of the dihydrofolate reductase (DHFR) inhibitor, methotrexate (MTX) to the medium in concentrations which completely inhibited pineal DHFR had little or no effect on pineal [BH4 + qBH2] whereas pinal [BH2 + B] and medium B increased up to 2-fold, suggesting that DHFR may be involved in the biosynthesis of BH4. A biopterin-bovine serum albumin conjugate is being used as an immunogen in rabbits to develop antibodies to biopterin, ultimately required for development of an immunoassay for biopterin.